Amyotrophic Lateral Sclerosis (ALS) is an incurable neurodegenerative disease characterized by death of upper and lower motor neurons. Familial ALS, a subtype of the disease in which a causative mutation is inherited, is linked in 1/5 of patients to mutations of the superoxide dismutase (SOD1) gene. Mice with a specific G93A mutation in SOD1 (mutant SOD1 or mSOD1) develop symptoms of ALS such as paralysis and death, and pathological findings of ALS such as motor neuron degeneration and astrogliosis. Using mSOD1 mouse models, recent studies have discovered that astrocytes, a supportive cell type under physiological conditions, become toxic to motor neurons as ALS progresses. In culture, mSOD1 motor neurons are viable, but mSOD1 or wild type motor neurons co-cultured with mSOD1 astrocytes will degenerate and die. Our lab became interested in this phenomenon of glial non-cell autonomous degeneration of motor neurons. We identified a protein complex, comprised of the actin binding protein ?-adducin and ion pump ?2-Na/K ATPase, which is overexpressed in mSOD1 astrocytes, mSOD1 mouse spinal cord at symptom onset and human patients with familial ALS. When the ?-adducin protein complex is knocked down in mSOD1 astrocytes, it prevents degeneration of co-cultured motor neurons. Moreover, when one allele of the ?2-Na/K ATPase is knocked out, mSOD1 mice have later onset of ALS symptoms and survive 10% longer. This demonstrated that the ?-adducin/?2-Na/K ATPase complex is worsening motor neuron degeneration in an ALS mouse model, but raises important questions such as which cell type is the complex functioning in in vivo to induce motor neuron degeneration, and why does mSOD1 mutation cause the complex to become toxic to motor neurons. This proposal addresses these two questions in the following way. First, mSOD1 mice will be bred with the ?2-Na/K ATPase knocked out in astrocytes and motor neurons specifically, to test the hypothesis that ?2-Na/K ATPase operates in astrocytes to induce non-cell autonomous degeneration of motor neurons. Next, ?-adducin will be mutated to forms that increase and decrease phosphorylation, to determine the functional significance of phosphorylation in the interaction and toxicity of the ?-adducin/?2-Na/K ATPase complex. Also, the role of phosphorylation in in vivo protein-protein interactions will be tested. Finally, the kinase responsible for phosphorylating ?-adducin, resulting in its toxicity to motor neurons, will be identified using rigorous biochemical and genetic methods. These experiments will provide mechanistic insights into glial-dependent neurodegeneration in ALS and related disorders, and potentially discover new therapeutic targets for the disease.